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We have used ELISA and culture of spiroplasmas in SP4 or M1A medium to detect Spiroplasma citri in citrus trees, periwinkles (Catharantus roseus) and leafhoppers in Iraq and Syria. On the basis of hundreds of analyses, we have found that the sensitivity of ELISA and culture are of the same order of magnitude. With citrus, both tests are able to detect S. citri in 95% of symptomatic nursery or field trees. The tests are not sensitive enough to reliably detect S. citri in symptomless trees. Therefore, a DNA hybridization technique was developed. The radioactive DNA probe was prepared either with total S. citri DNA or with the cloned DNA fragment carrying the spiralin gene of S. citri. Used in dot hybridization, the probe was able to detect concentrations of S. citri one 100th the size of those revealed by ELISA.

To study natural transmission of Spiroplasma citri in the Mediterranean area, periwinkles (Vinca rosea L.) were exposed to natural infection in several locations during the summer of 1983. Detection of S. citri in the periwinkles was assayed by ELISA and culture of the organisms. Some of the periwinkles developed yellows disease symptoms; they contained helical organisms in their sieve tubes, as determined by electron microscopy. A spiroplasma could be cultured from them, but their ELISA reaction for S. citri detection was negative. These results suggested that a spiroplasma different from S. citri had been discovered. The apparently new spiroplasma (P40) was examined for serological relationships with other spiroplasmas by metabolism inhibition and deformation tests. Spiroplasma P40 was found to have relatedness only to Group I spiroplasmas. Healthy periwinkles graft inoculated with shoots of the initial symptomatic periwinkle showed yellows symptoms 3 months after inoculation. Extracts of the initial and the graft-inoculated periwinkles gave positive ELISA reactions with anti-P40 IgG.

PPAV is a type of Mollicutes that was isolated in SP4 medium from seeds of apples affected by proliferation, a disease in which mycoplasma-like organisms (MLOs) are involved. However, PPAV is probably not the etiological MLO agent for at least two reasons: 1) optimal temperature growth is 43 C, and 2) in spite of numerous isolation attempts over several years, no second PPAV culture could be obtained. PPAV is surrounded by a single cytoplasmic membrane and forms typical fried egg-shaped colonies on solid medium. The organism grows in simplified mycoplasma media, such as BSR. In growth inhibition, it shows no serological relationships with any other mycoplasmas or acholeplasmas, including those cultured from the surfaces of plants. The absence of relatedness of PPAV to other Mollicutes was confirmed by DNA hybridization studies. The genome of PPAV is close to 10(9) daltons and contains 25.2 mol % G + C. Its genome size is similar to that of Acholeplasma spp. and Spiroplasma spp. It has, however, a clearcut sterol requirement and therefore cannot be an acholeplasma. Neither is it a spiroplasma since, though filamentous in BSR medium, it has never shown signs of helicity. Hence, PPAV is a taxonomical paradox.